Cell lines

Squamous cell carcinomas (SCC):A431 cells

Preparation Method

AZ1 was added to the medium of A431 cells for 48h

Reaction Conditions

15 μM, 48 h

Applications

USP25/28 inhibitor AZ1-mediated inhibition of USP28 led to changes as measured with predominant effects on pathways associated with cellular stress, cell cycle progression and DNA damage checkpoint and repair. An upregulation of DNA damage sensors like TP53BP1, MRE11 or RAD50 and simultaneously the downregulation of proteins involved in replication-coupled DNA repair, such as RAD51, RPA1 or RPA2. Of note, loss of USP28 activity was associated with a significant decrease of proteins involved in DNA replication.

Animal models

Usp25^+/+and Usp25^-/-mice(age-matched and sex-matched mice)

Preparation Method

At 12 weeks old, mice were injected with PBS or USP25/28 inhibitor AZ1 (40 mg per kg body weight) by gavage every 3 d for 8 weeks.

Dosage form

40 mg/kg, every 3 d for 8 weeks.

Applications

To test the effects of USP25/28 inhibitor AZ1 on DSS-induced colitis and found that USP25/28 inhibitor AZ1 gavage protected from weight loss and diarrhea and impaired colon shortening and potentiated the expression of proinflammatory cytokines and antibacterial peptides in colons.

USP25/28 inhibitor AZ1 (AZ1) is an orally active, selective, noncompetitive, dual ubiquitin specific protease (USP) 25/28 inhibitor with IC₅₀s of 0.7 μM and 0.6 μM, respectively. High expression levels of USP28 are essential for colon and breast cancers, and stabilization of c-Myc by USP28 is essential for tumor cell proliferation^[5]

USP25/28 inhibitor AZ1-mediated inhibition of USP28 led to changes as measured with predominant effects on pathways associated with cellular stress, cell cycle progression and DNA damage checkpoint and repair. An upregulation of DNA damage sensors like TP53BP1, MRE11 or RAD50 and simultaneously the downregulation of proteins involved in replication-coupled DNA repair, such as RAD51, RPA1 or RPA2^[3]

USP25/28 inhibitor AZ1 attenuates colitis and tumorigenesis in the mice mode^[1].To test the effects of AZ1 on DSS-induced colitis and found that AZ1 gavage protected from weight loss and diarrhea and impaired colon shortening and potentiated the expression of proinflammatory cytokines and antibacterial peptides. In addition, levels of p-p65, p-p38 and SOCS3 were potentiated and levels of TRAF3 and pSTAT3 were decreased in colon tissues from AZ1-treated mice. In addition, gavage of AZ1 maintained weight gain and reduced the bacteria count in feces after C. rodentium infection. inflammation and bacterial replication in the colon were impaired, expression of proinflammatory cytokines and antibacterial peptides was potentiated, levels of p-p65 and p-p38 were increased and levels of TRAF3 were decreased in colons of mice with AZ1 gavage^[2].Subchronic injection of AZ1 in 5 FAD mice markedly ameliorated memory deficits in cued FC and MWM tests.In addition, LTP impairment was reversed through long-term AZ1 administration.AZ1 administration attenuated microglial proliferation and activation in the hippocampusand cortex of 5×FAD mice.AZ1 ameliorated AD neuropathology by attenuating microglial activation^[4]

References:
[1]: Wrigley JD, Gavory G, et,al. Identification and Characterization of Dual Inhibitors of the USP25/28 Deubiquitinating Enzyme Subfamily. ACS Chem Biol. 2017 Dec 15;12(12):3113-3125. doi: 10.1021/acschembio.7b00334. Epub 2017 Nov 28. PMID: 29131570.
[2]: Wang XM, Yang C, et,al. The deubiquitinase USP25 supports colonic inflammation and bacterial infection and promotes colorectal cancer. Nat Cancer. 2020 Aug;1(8):811-825. doi: 10.1038/s43018-020-0089-4. Epub 2020 Jul 6. PMID: 35122046.
[3]: Prieto-Garcia C, Hartmann O, et,al. Inhibition of USP28 overcomes Cisplatin-resistance of squamous tumors by suppression of the Fanconi anemia pathway. Cell Death Differ. 2022 Mar;29(3):568-584. doi: 10.1038/s41418-021-00875-z. Epub 2021 Oct 5. PMID: 34611298; PMCID: PMC8901929.
[4]: Zheng Q, Li G, et,al. Trisomy 21-induced dysregulation of microglial homeostasis in Alzheimer's brains is mediated by USP25. Sci Adv. 2021 Jan 1;7(1):eabe1340. doi: 10.1126/sciadv.abe1340. PMID: 33523861; PMCID: PMC7775784.
[5]: Popov N, Wanzel M, et,al. The ubiquitin-specific protease USP28 is required for MYC stability. Nat Cell Biol. 2007 Jul;9(7):765-74. doi: 10.1038/ncb1601. Epub 2007 Jun 10. PMID: 17558397.